Minisatellite alterations in ZRT1 mutants occur via RAD52-dependent and RAD52-independent mechanisms in quiescent stationary phase yeast cells

Alterations in minisatellite DNA repeat tracts are associated with a variety of human diseases including Type 1 diabetes, progressive myoclonus epilepsy, and some types of cancer. However, in spite of their role in human health, the factors required for minisatellite alterations are not well understood. We previously identified a stationary phase specific increase in minisatellite instability caused by mutations in the high affinity zinc transporter ZRT1, using a minisatellite inserted into the ADE2 locus in Saccharomyces cerevisiae. Here, we examined ZRT1-mediated minisatellite instability in yeast strains lacking key recombination genes to determine the mechanisms by which these alterations occur. Our analysis revealed that minisatellite alterations in a Δzrt1 mutant occur by a combination of RAD52-dependent and RAD52-independent mechanisms. In this study, plasmid-based experiments demonstrate that ZRT1-mediated minisatellite alterations occur independently of chromosomal context or adenine auxotrophy, and confirmed the stationary phase timing of the events. To further examine the stationary phase specificity of ZRT1-mediated minisatellite alterations, we deleted ETR1 and POR1, genes that were previously shown to differentially affect the viability of quiescent or nonquiescent cells in stationary phase populations. These experiments revealed that minisatellite alterations in Δzrt1 mutants occur exclusively in quiescent stationary phase cells. Finally, we show that loss of ZRT1 stimulates alterations in a derivative of the human HRAS1 minisatellite. We propose that the mechanism of ZRT1-mediated minisatellite instability during quiescence is relevant to human cells, and thus, human disease.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.     

Hypermutable minisatellites,a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.     

Mouse DNA ''fingerprints'': analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains.

Human minisatellite probes cross-hybridize to mouse DNA and detect multiple variable loci. The resulting DNA "fingerprints" vary substantially between inbred strains but relatively little within an inbred strain. By studying the segregation of variable DNA fragments in BXD recombinant inbred strains of mice, at least 13 hypervariable loci were defined, 8 of which could be regionally assigned to mouse chromosomes. The assigned loci are autosomal, dispersed and not preferentially associated with centromeres or telomeres. One of these minisatellites is complex, with alleles 90 kb or more long and with internal restriction endonuclease cleavage sites which produce a minisatellite "haplotype" of multiple cosegregating fragments. In addition, one locus shows extreme germ-line instability and should provide a useful system for studying more directly the rates and processes of allelic variation of minisatellites.     

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.     

Novel Checkpoint Pathway Organization Promotes Genome Stability in Stationary-Phase Yeast Cells

Most DNA alterations occur during DNA replication in the S phase of the cell cycle. However, the majority of eukaryotic cells exist in a nondividing, quiescent state. Little is known about the factors involved in preventing DNA instability within this stationary-phase cell population. Previously, we utilized a unique assay system to identify mutations that increased minisatellite alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here we conducted a modified version of synthetic genetic array analysis to determine if checkpoint signaling components play a role in stabilizing minisatellites in stationary-phase yeast cells. Our results revealed that a subset of checkpoint components, specifically MRC1, CSM3, TOF1, DDC1, RAD17, MEC3, TEL1, MEC1, and RAD53, prevent stationary-phase minisatellite alterations within the quiescent cell subpopulation of stationary-phase cells. Pathway analysis revealed at least three pathways, with MRC1, CSM3, and TOF1 acting in a pathway independent of MEC1 and RAD53. Overall, our data indicate that some well-characterized checkpoint components maintain minisatellite stability in stationary-phase cells but are regulated differently in those cells than in actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element; rather, it may be loss of the checkpoint proteins'' other functions that contributes to DNA instability.     

Influences of array size and homogeneity on minisatellite mutation.

Unstable minisatellites display high frequencies of spontaneous gain and loss of repeats in the human germline. Most length changes arise through complex recombination events including intra-allelic duplications/deletions and inter-allelic transfers of repeats. Definition of the factors modulating instability requires both measurement of mutation rate and detailed analysis of mutant structures at the level of individual alleles. We have measured mutation rates in sperm for a wide range of alleles of the highly unstable human minisatellite CEB1. Instability varies by three orders of magnitude between alleles and increases steadily with the size of the tandem array. Structural analysis of mutant molecules derived from six alleles revealed that it is the rate of intra-allelic rearrangements which increases with array size and that intra-allelic duplication events tend to cluster within homogeneous segments of alleles; both phenomena resemble features of trinucleotide repeat instability. In contrast, inter-allelic transfers occur at a fairly constant rate, irrespective of array length, and show a mild polarity towards one end of the minisatellite, suggesting the possible influence of flanking DNA on these conversion-like events.     

Recombination analysis of the human minisatellite MsH42 suggests the existence of two distinct pathways for initiation and resolution of recombination at MsH42 in rat testes nuclear extracts

We have previously described a GC-rich human minisatellite, termed MsH42, which exists in two allelic forms, long and short. Here, we have identified a third allele of medium length and localized the MsH42 locus in the chromosome 15q25.1 inside an intron belonging to a gene of unknown function. The recombinogenic potential of the three alleles was assayed in vitro incubating pBR322-based constructs containing two copies of the minisatellite MsH42 with its flanking sequences, in the presence of rat testes nuclear extracts. This assay system was configured to monitor only reciprocal exchange type events and not gene conversion. All MsH42 allelic sequences enhanced intramolecular homologous recombination promoting high rates (approximately 76%) of equal crossover, the long allele showing the highest recombinogenic activity. Removal of the MsH42 long allele flanking sequences, which are identical in the three alleles, provoked a decrease in the enhancement of recombination and in the frequency of equal crossovers, suggesting that these sequences are important for the recombinogenic activity and for the correct pairing between homologous sequences. The occurrence of some complex recombination events within the minisatellite MsH42 suggests the existence of processes related to polymerase slippage and unwinding with reinvasion during the repair synthesis. Our findings point toward the existence of two distinct biochemical pathways for initiation and resolution of recombination at the minisatellite MsH42. Finally, the in vitro recombination system employed in this study could provide an approach to dissect processes of repetitive DNA instability and recombination.     

Minisatellite DNA fingerprints of salmonid fishes

The human minisatellite probes 33.6 and 33.15 cross-hybridized to DNA digests of Atlantic salmon, brown trout and rainbow trout revealing complex multi-banded patterns. These DNA fingerprints (in excess of 40 resolvable fragments in some cases) were highly polymorphic, individual specific and found to be stable, both somatically and in the germline. Pedigree analysis of an Atlantic salmon family confirmed that the minisatellite fragments showed Mendelian inheritance. With only a single occurrence of linkage and allelism being observed it is likely the minisatellite loci are widely distributed throughout the salmonid genome. The potential applications for both multi- and single locus minisatellite probes in salmonid research are discussed.     

Length and sequence heterozygosity differentially affect HRAS1 minisatellite stability during meiosis in yeast

Minisatellites, one of the major classes of repetitive DNA sequences in eukaryotic genomes, are stable in somatic cells but destabilize during meiosis. We previously established a yeast model system by inserting the human Ha-ras/HRAS1 minisatellite into the HIS4 promoter and demonstrated that our system recapitulates all of the phenotypes associated with the human minisatellite. Here we demonstrate that meiotic minisatellite tract-length changes are half as frequent in diploid cells harboring heterozygous HRAS1 minisatellite tracts in which the two tracts differ by only two bases when compared to a strain with homozygous minisatellite tracts. Further, this decrease in alteration frequency is entirely dependent on DNA mismatch repair. In contrast, in a diploid strain containing heterozygous minisatellite tract alleles differing in length by three complete repeats, length alterations are observed at twice the frequency seen in a strain with homozygous tracts. Alterations consist of previously undetectable gene conversion events, plus nonparental length alteration events seen previously in strains with homozygous tracts. A strain containing tracts with both base and length heterozygosity exhibits the same level of alteration as a strain containing only length heterozygosity, indicating that base heterozygosity-dependent tract stabilization does not affect tract-length alterations occurring by gene conversion.     

Expansions and contractions in 36-bp minisatellites by gene conversion in yeast

The instability of simple tandem repeats, such as human minisatellite loci, has been suggested to arise by gene conversions. In Saccharomyces cerevisiae, a double-strand break (DSB) was created by the HO endonuclease so that DNA polymerases associated with gap repair must traverse an artificial minisatellite of perfect 36-bp repeats or a yeast Y' minisatellite containing diverged 36-bp repeats. Gene conversions are frequently accompanied by changes in repeat number when the template contains perfect repeats. When the ends of the DSB have nonhomologous tails of 47 and 70 nucleotides that must be removed before repair DNA synthesis can begin, 16% of gene conversions had rearrangements, most of which were contractions, almost always in the recipient locus. When efficient removal of nonhomologous tails was prevented in rad1 and msh2 strains, repair was reduced 10-fold, but among survivors there was a 10-fold reduction in contractions. Half the remaining events were expansions. A similar decrease in the contraction rate was observed when the template was modified so that DSB ends were homologous to the template; and here, too, half of the remaining rearrangements were expansions. In this case, efficient repair does not require RAD1 and MSH2, consistent with our previous observations. In addition, without nonhomologous DSB ends, msh2 and rad1 mutations did not affect the frequency or the distribution of rearrangements. We conclude that the presence of nonhomologous ends alters the mechanism of DSB repair, likely through early recruitment of repair proteins including Msh2p and Rad1p, resulting in more frequent contractions of repeated sequences.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

Radiation-induced genomic instability in tandem repeat sequences is not predictive of unique sequence instability

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear gamma-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.     

Identification of hypervariable single locus minisatellite DNA probes in the blue tit Parus caeruleus

We report the isolation of a set of hypervariable minisatellite DNA sequences from a blue tit Parus caeruleus genomic DNA library. In our strategy, we cloned a minisatellite-rich DNA fraction into a charomid vector. The resulting cosmid library was screened with the two minisatellite DNA probes 33.6 and 33.15 for recombinants containing a minisatellite DNA insert. A total of 233 positive clones were isolated. Of 37 clones that have been analysed, nine gave polymorphic signals and can be used as single locus probes (SLPs). Four of the SLPs were investigated in more detail. The number of alleles, the heterozygosity and the mutation rate were estimated. Linkage analysis revealed that two of these loci were linked. The SLPs are of value to studies of the mating system and reproductive success in the blue tit, and may also be useful in population genetic studies.     

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.